Microencapsulation of bacteriophages and related products

ABSTRACT

An encapsulated bacteriophage formulation and a method for encapsulating bacteriophages and bacteriophage-related products in polymeric microcapsules is provided. Some embodiments of the method of producing the encapsulated bacteriophages involves a water-in-oil-in-water double emulsion.

FIELD OF THE INVENTION

The present invention relates to the general field of bacteriophages andis more particularly concerned with microencapsulation of bacteriophagesand related products.

BACKGROUND

Multidrug and pandrug-resistant bacteria pose a major threat to human,animal and plant health. Multidrug resistant strains of bacteria such asStaphylococcus aureus and Pseudomonas aeruginosa play a prominent roleas an etiological agent of serious nosocomial and community acquiredinfections. These infections often lead to bacteremia, sepsis, and highrate of mortality and morbidity.

Hence, the development of new therapeutic and prophylactic strategiesfor the control of bacterial infections in human patients, livestock,and produce is needed.

An alternative or supplement to antibiotic therapy is the use ofbacteriophages to target bacterial infections. Advantages of phagetherapy resides in the bactericidal effect of phages, their auto-dosingability by proliferating locally in areas infected by their bacterialhost, their low inherent toxicity, their minimal disruption of thenormal bacterial flora and microbiome, their synergy with antibiotics,the cocktail formulation versatility and their ability to clearbiofilms. Reports of successful bacteriophage therapy have beenextensively reviewed. Clinical use of bacteriophages often involve thedelivery of naked phages in simple liquid formulations. Theeffectiveness of phage therapy can be affected by multiple factors suchas lack of specificity of the phage to the target infection, rapidclearance by the immune system, inactivation by unfavorableenvironmental factors such as adverse enzymes, pH, and temperature andlong term storage. In view of this, administration of phages in humanand animals requires an appropriate delivery system.

Although a number of advanced methods for controlled and or targeteddrug delivery have been trying to build on the conventional drugdelivery method of microencapsulation proposed in the 1960s, thesetechniques have not addressed the growing need of encapsulatingbacteriophages with maximal efficiency in biodegradable polymericmicrocapsules. For example, liposomes are not suited for deliveringphages are they are thermodynamically unstable, and tend to fuse,resulting in the early release of the therapeutic agent. Phages havealso been encapsulated in alginate, chitosan, and pectin, (withmitigated results) with loss of lytic activity of the phages and suchformulations do not provide sufficient protection again stomach acidity,and do not show long term stability of the formulations. Moreover,phages have been encapsulated in poly (DL-lactic co-glycolic acid)(PGLA), but this resulted in loss of stability and integrity of thebacteriophages after 7 days due to the physical interaction at thesurface or encapsulation within the PGLA porous matrix.

Accordingly, there exists a need for a new ways to encapsulatebacteriophages.

SUMMARY OF THE INVENTION

Novel amino acid based biopolymers are easy to produce, have low cost,maintain phage viability, are stable, do not cause immunogenicreactions, are biodegradable, allow for high phage loading and have aproper phage release rate.

Incorporating phages into biodegradable amino acid-based microparticleswould be beneficial for many reasons, including protection fromdenaturation and inactivation, as well as enhanced therapeutic activitythat prolong the release and presence of the phage in the human body.Other advantages of encapsulating phages into microparticles reside inincreased circulation time of phages, decreased systemic clearance,protection from enzymatic or acidic degradation, and controlled release.

The present invention proposes methods for encapsulating bacteriophagesand compositions prepared using such methods. Possible uses for theinvention include local delivery on skin, ears, throat, nose, pulmonarydelivery, mucosal membranes, vagina, burns, ulcers (pressure, venous,diabetic, etc.), surgical wounds, other types of wounds. Are alsocontemplated intramuscular delivery, subcutaneous deliver, oral orintraoeritonial delivery. The proposed compositions are also usable forreducing pathogen colonization in livestock, biocontrol of raw meats andfresh products, biopreservation and expansion of shelf life of readyproducts.

The present invention relates to encapsulating bacteriophages intopolymeric microparticles. In particular this invention pertains toencapsulating bacteriophage, bacteriophage products, or phage-relatedproducts, such as endolysins, lysostaphins, phage proteins or phageenzymatic formulations and the methods of preparing compositionsincorporating these encapsulations. The water-based formulations providea mean for the controlled release of bacteriophages for differentbacterial targets.

The invention is based on encapsulated phages in microcapsules made of asuitable polymer, such as a Polyester amide urea (PEAU), a leucine-basedpoly ester amide polymer, or another amino acid based copolymer. Due toboth groups, ester and amide, such polymers are biodegradable (estergroup) and have good thermal stability and mechanical strength (amidegroup with strong intermolecular interactions). The incorporation ofleucine, or other suitable amino acid, improves the biocompatibility ofthe polymer. The biodegradation rate of this polymer can easily beadjusted by changing its exact composition and molecular weight. Whenmicrocapsules are formed, the liberation rate of any productincorporated therein can be adjusted by controlling the size andthickness of the microcapsules.

Such a polymer is synthesized, in some embodiments, by interfacialpolycondensation of the monomer L6, di-p-sulfonic acid salt ofbis-(L-leucine)-1,6-hexylene diester with trisphogene/sebacoyl chloridewith water/dichloromethane system. The use of dichloromethane allowsdirect utilization of the biocomposite for phages incorporationtherefore for microcapsules fabrication. This method is fast,irreversible, involves two immiscible phases at room temperature andlead to high molecular weight polymer. Synthesis of the monomer L6 wasexecuted in the presence of p-toluene sulfonic acid by condensation ofL-leucine with 1,6-hexanediol in refluxed cyclohexane, because it isless toxic than solvents such as benzene. Purification includesrecrystallization from water, filtration and drying under vacuum.

The formulations containing microcapsules are fabricated using awater-in-oil-in-water double emulsion-solvent, where the addition of thebacteriophages occurs in some embodiments in the secondary emulsion tominimize their exposure with the solvent dichloromethane (DCM). The DCMcan also be replaced by an other suitable solvent, such as ethylacetate, chloroform, or another organic solvent. Bacteriophages arestable against DCM, especially during the time of the reaction. It wasfound that, in some embodiments, there is no need to use a hardeningtank during preparation of the microcapsules. Hardening tanks requiredilution of the microcapsule preparation, for example by a factor of 5or more. In addition to requiring additional processes to recover themicrocapsules in the relatively large volume of liquid, use of hardeningtank results in dilution of any component left in the aqueous phase inwhich the microcapsules are suspended, such as bacteriophages.

Other polymers usable in the invention include:

A polymer selected from

(1) a poly (ester amide urea) wherein at least one diol, at least onediacid, and at least one amino acid are linked together through an esterbond, an amide bond, and a urea bond,

(2) a poly (ester urethane urea) wherein at least one diol and at leastone amino acid are linked together through an ester bond, a urethanebond, and a urea bond,

(3) a poly (ester amide urethane urea) wherein at least one diol, atleast one diacid, and at least one amino acid are linked togetherthrough an ester bond, an amide bond, a urethane bond, and a urea bond,

(4) a poly (ester amide urethane) wherein at least one diol, at leastone diacid, and at least one amino acid are linked together through anester bond, an amide bond, and a urethane bond,

(5) a poly (ester urea) wherein at least one diol and at least one aminoacid are linked together through an ester bond and a urea bond, and

(6) a poly (ester urethane) wherein at least one diol and at least oneamino acid are linked together through an ester bond and a urethanebond,

further wherein

the at least one diol is a compound of formula:

HO—R₁—OH, R₁ is chosen from C₂-C₁₂ alkylene optionally interrupted by atleast one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

the at least one diacid is a compound of formula:

HO—(CO)—R₃—(CO)—OH, R₃ is C₂-C₁₂ alkylene,

the at least one amino acid is chosen from naturally occurring aminoacids and non-naturally occurring amino acid.

In some embodiments, the polymer is selected from

(1) a poly (ester amide urea) wherein at least one diol, at least onediacid, and at least one amino acid are linked together through an esterbond, an amide bond, and a urea bond,(2) a poly (ester urethane urea) wherein at least one diol and at leastone amino acid are linked together through an ester bond, a urethanebond, and a urea bond,(3) a poly (ester amide urethane urea) wherein at least one diol, atleast one diacid, and at least one amino acid are linked togetherthrough an ester bond, an amide bond, a urethane bond, and a urea bond,and(4) a poly (ester amide urethane) wherein at least one diol, at leastone diacid, and at least one amino acid are linked together through anester bond, an amide bond, and a urethane bond,wherein the at least one diol, at least one diacid, and at least oneamino acid are as defined in claim 1.

In some more specific embodiments of the invention, the polymer is apoly (ester amide urea) comprising the following two blocks with randomdistribution thereof:

wherein

the ratio of l:m ranges from 0.05:0.95 to 0.95:0.05, l+m=1,

R₁ is chosen from C₂-C₁₂ alkylenes optionally interrupted by at leastone oxygen, C₃-C₈ cycloalkylenes, C₃-C₁₀ cycloalkylalkylenes,

R₃ is C₂-C₁₂ alkylene,R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids so that the carbon to which R₂ or R₄ is attached has L orD chirality.

In some more specific embodiments of the invention, the polymer is poly(ester urethane urea) comprising the following two blocks with randomdistribution thereof:

whereinthe ratio of l:m ranges from 0.05:0.95 to 0.95:0.05, l+m=1,R₁ and R₅ are independently chosen from C₂-C₁₂ alkylenes optionallyinterrupted by at least one oxygen, C₃-C₈ cycloalkylenes, C₃-C₁₀cycloalkylalkylenes,

andR₂ and R₄ are independently chosen from the side chains of L- andD-amino acids so that the carbon to which R₂ or R₄ is attached has L orD chirality.

In some more specific embodiments of the invention, the polymer is poly(ester amide urethane urea) comprising the following three blocks withrandom distribution thereof:

whereinthe ratio of l:m:k ranges from 0.05:0.05:0.90 to 0.90:0.05:0.05,l+m+k=1,R₁ and R₅ are independently chosen from C₂-C₁₂ alkylenes optionallyinterrupted by at least one oxygen, C₃-C₈ cycloalkylenes, C₃-C₁₀cycloalkylalkylenes,

R₃ is C₂-C₁₂ alkylene, andR₂ and R₄ are independently chosen from the side chains of L- andD-amino acids so that the carbon to which R₂ or R₄ is attached has L orD chirality.

In some more specific embodiments of the invention, the polymer is(ester amide urethane) comprising the following two blocks with randomdistribution thereof:

wherein

the ratio of l:m ranges from 0.05:0.95 to 0.95:0.05, l+m=1,

R₁ and R₅ are independently chosen from C₂-C₁₂ alkylenes optionallyinterrupted by at least one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀cycloalkylalkylene,

R₃ is C₂-C₁₂ alkylene, andR₂ and R₄ are the same and selected from the side chains of L- andD-amino acids so that the carbon to which R₂ or R₄ is attached has L orD chirality.

In the above polymers, in some very specific embodiments of theinvention, one or more of the following hold: R₁ is —(CH₂)₆—, R₃ is—(CH₂)₈—, or both R₂ and R₄ are the side chain of L-leucine.

Blends of the above-mentioned polymers are also usable in thepreparation of the compositions of the present invention. More detailsregarding such polymers and others usable with the present invention areprovided in PCT application PCT/US2016/038527 and U.S. patentapplication Ser. No. 15/188,783, the contents of which is herebyincorporated by reference in its entirety. The present applicationclaims priority from U.S. provisional patent application 62/353,658filed Jun. 23, 2016, U.S. patent application Ser. No. 16/310,732 filedDec. 17, 2018 and U.S. patent application Ser. No. 17/088,899 filed Nov.4, 2020, the contents of which is hereby incorporated by reference inits entirety.

In a broad aspect, the invention provides a composition including:polymer microcapsules; at least one of active bacteriophages andbacteriophage related products encapsulated in the polymermicrocapsules; wherein the polymer microcapsules include an amino-acidbased polymer.

The invention may also provide a composition wherein activebacteriophages are encapsulated in the polymer microcapsules.

The invention may also provide a composition wherein the activebacteriophages are in a first aqueous suspension in the polymermicrocapsules.

The invention may also provide a composition wherein the first aqueoussuspension includes polyvinyl alcohol.

The invention may also provide a composition wherein the first aqueoussuspension includes between 0.1% and 10% w/v of the polyvinyl alcohol.

The invention may also provide a composition wherein the polyvinylalcohol has a mean molecular weight of between 10 kDa and 400 kDa.

The invention may also provide a composition wherein the polyvinylalcohol has a mean molecular weight of between 65 kDa and 90 kDa.

The invention may also provide a composition wherein the polyvinylalcohol has a mean molecular weight of between 10 kDa and 35 kDa.

The invention may also provide a composition wherein the polymermicrocapsules are in a second aqueous suspension.

The invention may also provide a composition wherein the second aqueoussuspension also includes polyvinyl alcohol.

The invention may also provide a composition wherein the second aqueoussuspension includes between 1% and 5% w/v of the polyvinyl alcohol.

The invention may also provide a composition wherein the second aqueoussuspension includes between 2.5% and 5% w/v of the polyvinyl alcohol.

The invention may also provide a composition wherein the polyvinylalcohol is in a higher concentration in the second aqueous solution thanin the first aqueous solution.

The invention may also provide a composition wherein the polymermicrocapsules are in a second aqueous suspension.

The invention may also provide a composition wherein the activebacteriophages are adsorbed on inorganic particles encapsulated in thepolymer microcapsules.

The invention may also provide a composition wherein the bacteriophagerelated products are adsorbed on inorganic particles encapsulated in thepolymer microcapsules.

The invention may also provide a composition wherein the inorganicparticles include particles of at least one salt selected from the groupconsisting of CaCO₃, Ca₃(PO₄)₂, MgCO₃, and Mg₃(PO₄)₂.

The invention may also provide a composition wherein the inorganicparticles have a mean size between 2 μm and 15 μm.

The invention may also provide a composition wherein the inorganicparticles have a mean size between 2 μm and 4 μm.

The invention may also provide a composition wherein the polymermicrocapsules have a mean size between 20 μm and 100 μm.

The invention may also provide a composition wherein the polymermicrocapsules have a mean size between 20 μm and 50 μm.

The invention may also provide a composition wherein the polymermicrocapsules have an upper limit size of 250 μm or less.

The invention may also provide a composition wherein the microcapsulesare hollow and wherein a thickness of the polymer in the microcapsulesis between 3% and 15% of the mean size.

The invention may also provide a composition wherein the polymermicrocapsules have an upper limit size of 100 μm or less.

The invention may also provide a composition wherein the bacteriophagerelated products are selected from the group consisting of endolysins,lysostaphins, phage proteins, phage enzymatic formulations, andcombinations thereof.

The invention may also provide a composition wherein the amino-acidbased polymer is selected from

(1) a poly (ester amide urea) wherein at least one diol, at least onediacid, and at least one amino acid are linked together through an esterbond, an amide bond, and a urea bond,

(2) a poly (ester urethane urea) wherein at least one diol and at leastone amino acid are linked together through an ester bond, a urethanebond, and a urea bond,

(3) a poly (ester amide urethane urea) wherein at least one diol, atleast one diacid, and at least one amino acid are linked togetherthrough an ester bond, an amide bond, a urethane bond, and a urea bond,

(4) a poly (ester amide urethane) wherein at least one diol, at leastone diacid, and at least one amino acid are linked together through anester bond, an amide bond, and a urethane bond,

(5) a poly (ester urea) wherein at least one diol and at least one aminoacid are linked together through an ester bond and a urea bond, and

(6) a poly (ester urethane) wherein at least one diol and at least oneamino acid are linked together through an ester bond and a urethanebond,

further wherein

-   -   the at least one diol is a compound of formula:        HO—R₁—OH, R₁ is chosen from C₂-C₁₂ alkylene optionally        interrupted by at least one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀        cycloalkylalkylene,

-   -   the at least one diacid is a compound of formula:

HO—(CO)—R₃—(CO)—OH, R₃ is C₂-C₁₂ alkylene,

-   -   the at least one amino acid is chosen from naturally occurring        amino acids and non-naturally occurring amino acid.

The invention may also provide a composition wherein the amino-acidbased polymer is a poly (ester amide urea) comprising the following twoblocks with random distribution thereof:

whereinthe ratio of l:m ranges from 0.05:0.95 to 0.95:0.05, l+m=1,R₁ is chosen from C₂-C₁₂ alkylenes optionally interrupted by at leastone oxygen, C₃-C₈ cycloalkylenes, C₃-C₁₀ cycloalkylalkylenes,

R₃ is alkylene,R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids so that the carbon to which R₂ or R₄ is attached has L orD chirality.

The invention may also provide a composition wherein the ratio of l:mranges from 0.25:0.75 to 0.75:0.25, l+m=1.

The invention may also provide a composition wherein R₁ is —(CH₂)₆—, R₃is —(CH₂)₈— and both R₂ and R₄ are the side chain of L-leucine.

The invention may also provide a composition wherein the amino-acidbased polymer has a polydispersity of 1.15 or less.

The invention may also provide a composition wherein the amino-acidbased polymer has a molecular weight between 15 kDa and 30 kDa.

The invention may also provide a composition wherein the amino-acidbased polymer is amorphous.

The invention may also provide a composition wherein the activebacteriophages include at least two different strains of bacteriophages.

The invention may also provide a composition wherein the at least twodifferent strains of bacteriophages include strains of bacteriophagesfrom more than one family.

The invention may also provide a composition wherein the composition isliquid form and has a viscosity small enough to allow pulverizationthrough a nozzle.

The invention may also provide a composition wherein the composition ispowder form.

The invention may also provide a composition wherein the composition isin liquid form, and the polymer microcapsules are suspended in asolution including a poloxamer.

The invention may also provide a composition wherein the poloxamer ispoloxamer 407.

The invention may also provide a composition wherein the poloxamer is ina concentration of between 10 and 30 percent.

The invention may also provide a composition wherein the poloxamer has amean molecular weight of between 9500 kDa and 15000 kDa.

The invention may also provide a composition wherein the composition isin gel form.

The invention may also provide a composition wherein the polymermicrocapsules contain on average more than 4 active bacteriophages.

The invention may also provide a composition wherein the polymermicrocapsules contain on average more than 100 active bacteriophages.

The invention may also provide a composition wherein the compositionfurther includes active bacteriophages outside of the polymermicrocapsules.

The invention may also provide a composition further including a drugselected from the set consisting of antibiotics, pain killer andhemostatic drug.

The invention may also provide a composition wherein the compositioncomprises active bacteriophages, and wherein the composition has astability such that at least 10% or at least 1% of the active phagesremain active after storage of the composition for one year at 4° C.

The invention may also provide a composition wherein at least some ofthe bacteriophages are dispersed in the amino-acid based polymer.

In another broad aspect, the invention provides a method for preparing acomposition including polymer microcapsules in which at least one ofactive bacteriophages and bacteriophage related products areencapsulated in the polymer microcapsules, the method including:preparing a first aqueous phase; preparing a second aqueous phaseincluding the at least one of active bacteriophages and bacteriophagerelated products suspended therein; preparing an hydrophobic phaseincluding an amino-acid based polymer dissolved therein; emulsifying thefirst aqueous phase in the hydrophobic phase to prepare a primaryemulsion; and emulsifying the primary emulsion in the second aqueousphase to prepare a secondary emulsion.

The invention may also provide a method wherein the primary emulsionincludes the first aqueous phase and the hydrophobic phase in a ratio offrom 1:2 to 1:20.

The invention may also provide a method wherein the primary emulsionincludes the first aqueous phase and the hydrophobic phase in a ratio ofabout 1:10.

The invention may also provide a method wherein the secondary emulsionincludes the primary emulsion and the second aqueous phase in a ratio offrom 1:2 to 1:20.

The invention may also provide a method wherein the secondary emulsionincludes the primary emulsion and the second aqueous phase in a ratio offrom 1:2 to 1:5.

The invention may also provide a method further including evaporatingthe organic solvent.

The invention may also provide a method wherein the organic solvent isevaporated without transfer to a hardening tank.

The invention may also provide a method wherein the organic solventincludes at least one of dichloromethane (DCM), ethylacetate andchloroform.

The invention may also provide a method wherein the organic solventincludes dichloromethane (DCM).

The invention may also provide a method wherein emulsifying the firstaqueous phase in the hydrophobic phase to prepare the primary emulsionincludes stirring the first aqueous phase in the hydrophobic phase usingan homogenizer operating a between 8000 RPM and 50000 RPM duringpreparation of the primary emulsion.

The invention may also provide a method wherein the homogenizer operatesat 10000 RPM or less during preparation of the primary emulsion.

The invention may also provide a method wherein the homogenization ofthe primary emulsion lasts for 30 s or less.

The invention may also provide a method wherein emulsifying thehydrophobic phase in the second aqueous phase to prepare the secondaryemulsion includes adding the primary emulsion dropwise to the secondaqueous phase while stirring the second aqueous phase using ahomogenizer operating a between 8000 RPM and 25000 RPM.

The invention may also provide a method wherein the homogenizer operatesat 10000 RPM or less during preparation of the secondary emulsion.

The invention may also provide a method wherein homogenization lastsless than 30 s during preparation of the secondary emulsion.

The invention may also provide a method wherein homogenization lastsless than 10 s during preparation of the secondary emulsion.

The invention may also provide a method wherein emulsifying thehydrophobic phase in the second aqueous phase to prepare the secondaryemulsion includes adding the primary emulsion to the second aqueousphase while stirring the second aqueous phase at between 100 and 1000RPM.

The invention may also provide a method wherein the primary emulsion isadded dropwise to the second aqueous phase.

The invention may also provide a method wherein stirring is performedusing an object plunged in the second aqueous phase.

The invention may also provide a method wherein the object is a magneticbar rotated by a magnetic stirrer.

The invention may also provide a method wherein stirring is performed atbetween 200 RPM and 600 RPM.

The invention may also provide a method wherein the hydrophobic phaseincludes from 5% to 20% w/v of polymer.

The invention may also provide a method wherein the hydrophobic phaseincludes about 13% w/v of polymer.

The invention may also provide a method further including dialysing thesecondary emulsion.

The invention may also provide a method wherein the first aqueous phaseincludes Kolliphor P188.

The invention may also provide a method wherein the first aqueous phaseincludes polyvinyl alcohol.

The invention may also provide a method wherein the first aqueous phaseincludes between 1% and 5% w/v of the polyvinyl alcohol and thepolyvinyl alcohol has a mean molecular weight of between 10 kDa and 100kDa.

The invention may also provide a method wherein the second aqueous phasealso includes polyvinyl alcohol.

The invention may also provide a method wherein the second aqueous phaseincludes between 1% and 5% w/v of the polyvinyl alcohol and thepolyvinyl alcohol has a mean molecular weight of between 10 kDa and 100kDa.

The invention may also provide a method wherein the bacteriophagerelated products are selected from the group consisting of endolysins,lysostaphins, phage proteins, phage enzymatic formulations, andcombinations thereof.

The invention may also provide a method wherein the amino-acid basedpolymer is any of the polymers selected from

(1) a poly (ester amide urea) wherein at least one diol, at least onediacid, and at least one amino acid are linked together through an esterbond, an amide bond, and a urea bond,

(2) a poly (ester urethane urea) wherein at least one diol and at leastone amino acid are linked together through an ester bond, a urethanebond, and a urea bond,

(3) a poly (ester amide urethane urea) wherein at least one diol, atleast one diacid, and at least one amino acid are linked togetherthrough an ester bond, an amide bond, a urethane bond, and a urea bond,

(4) a poly (ester amide urethane) wherein at least one diol, at leastone diacid, and at least one amino acid are linked together through anester bond, an amide bond, and a urethane bond,

(5) a poly (ester urea) wherein at least one diol and at least one aminoacid are linked together through an ester bond and a urea bond, and

(6) a poly (ester urethane) wherein at least one diol and at least oneamino acid are linked together through an ester bond and a urethanebond,

further wherein

-   -   the at least one diol is a compound of formula:        HO—R₁—OH, R₁ is chosen from C₂-C₁₂ alkylene optionally        interrupted by at least one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀        cycloalkylalkylene,

-   -   the at least one diacid is a compound of formula:

HO—(CO)—R₃—(CO)—OH, R₃ is C₂-C₁₂ alkylene,

-   -   the at least one amino acid is chosen from naturally occurring        amino acids and non-naturally occurring amino acid.

The invention may also provide a method wherein amino-acid based polymeris a poly (ester amide urea) comprising the following two blocks withrandom distribution thereof:

whereinthe ratio of l:m ranges from 0.05:0.95 to 0.95:0.05, l+m=1,R₁ is chosen from C₂-C₁₂ alkylenes optionally interrupted by at leastone oxygen, C₃-C₈ cycloalkylenes, C₃-C₁₀ cycloalkylalkylenes,

R₃ is C₂-C₁₂ alkylene,R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids so that the carbon to which R₂ or R₄ is attached has L orD chirality.

The invention may also provide a method wherein the ratio of l:m rangesfrom 0.25:0.75 to 0.75:0.25, l+m=1.

The invention may also provide a method wherein R₁ is —(CH₂)₆—, R₃ is—(CH₂)₈— and both R₂ and R₄ are the side chain of L-leucine.

The invention may also provide a method wherein the amino-acid basedpolymer has a molecular weight between 15 kDa and 30 kDa.

The invention may also provide a method further including dissolvingpolyvinyl alcohol in the secondary emulsion after formation of thesecondary emulsion.

The invention may also provide a method further including dissolving agelling agent in the secondary emulsion.

The invention may also provide a method wherein the gelling agent ispoloxamer 407.

The invention may also provide a method wherein the gelling agent is apoloxamer having a mean molecular weight of between 9500 kDa and 15000kDa.

The invention may also provide a method wherein the second aqueous phaseincludes the active bacteriophages at between 10⁷ and 10¹⁴ PFU/mL.

Advantageously, in some embodiments, the present invention allowsformation of encapsulating polymer microcapsules that are large enough(for example several tens of microns or more) to contain a usefulquantity of bacteriophages. In addition, amino acid based biodegradablepolymers (AABBPs) for medical applications are superior to the currentlycommercialized polyesters. They possess vast availability of variablebuilding blocks, which makes it possible to synthesize variouscopolymers containing fragments of different classes, allowing to designbiodegradable polymeric biomaterials with a widely tunablephysical-chemical, biochemical and mechanical properties—fromviscous-flow and elastic to high-strength materials.

The polymers chosen in the present patent application have NH—CO bondsthat provide a high affinity with tissues. In some embodiments, afterbiodegradation the AABBPs release weaker acidic products in much lowerquantities as compared with poly-α-hydroxy acids, thus increasing theirbiocompatibility with mammalian/human cells. For example, one of thebuilding blocks of PEAU, PEAs release neutral (zwitterionic) amino acidsand diols, and relatively week fatty diacids (e.g. adipic acid with pKa4.43 and 5.41) after its final hydrolysis. The other building block ofPEAU, PEU, releases normal metabolites such as CO2 and amino acids, andneutral and readily metabolized diols, following degradation.

Another advantage for microencapsulation and smooth biodegradation isthat these AABBPs are, in some embodiments, amorphous, or have asemi-crystalline structure that has a unique property of becomingamorphous and retaining its structure after melting/cooling cycle.

AABBPs possess typically much higher shelf life compared topoly(lactic/glycolic acid) polymers because as polycondensation typepolymers they do not subject depolymerization in storage.

For orthopedic and medical device applications, the biodegradablepolymer obtained on the basis of the PEU composed of L-phenylalanine and1,6-hexanediol, has a Young's modulus up to 6.1±1.1 GPa. These are thefirst degradable polymeric constructs with moduli in the range of 6.0GPa, which is substantially higher than the moduli of other,commercially available and widely used polyesters including tyrosinederived polycarbonates (≈1-2 GPa), poly(L-lactic acid), PLLA (≈3-3.5GPa), and poly(propylene fumarate) (2.2 GPa) used in numerousregenerative medicine and orthopedic applications. The obtainedmechanical and biochemical data show that the new PEU materials may beapt to provide structural support and facilitate new tissue regenerationstrategies in load bearing applications.

Another significant limitation of polyesters, such as polycaprolactoneand PLLA, is that they degrade very slowly. It can take years for thesematerials to degrade and resorb fully, and the associated acidificationthat results often leads to inflammation. Contrariwise, thebiodegradation of PEUs does not typically cause a local acidicenvironment causing inflammation to build up. During melting assays, theTg of PEU remained the same with no melting peak observed, indicatingthat no crystallinity was present. This provides relatively quick andcomplete biodegradation in contrast to e.g. PLLA in which the highinitial crystallinity form large amounts of tiny and rough highlyresistant particles when the amorphous phase was degraded. Suchparticles are now known as very inflammatory and thus well explain thelate dramatic inflammatory response observed in many studies.

Advantageously, PVA had been found to be an excellent surfactant forpreparing microcapsules by W/O/W double emulsion method. PVAs of variousmolecular weights (31,000 and 84,000-89,000 Da) were used, and very goodmicrocapsules (in terms of sized and size distribution) were fabricated,but other molecular weights are also usable. High-molecular-weight PVAforms microcapsules of desired size, and allows for the formation of anelastic films after evaporation of water, thus, it is of high interestfor preparing various types of bacteriophage containing film-like wounddressings. Adherence of the microcapsules suspension to the wound sitecan be optimized by the addion of a high-molecular-weight PVA(84,000-89,000 Da) to the microcapsule suspension, which will allow forthe formation of an elastic film after the evaporation of water content.Water-based spray dressings are promising since organic solvents likechloroform and dichloromethane are prohibited for medical uses, andalthough ethanol has been approved for medical applications, itinactivates bacteriophages. Advantages of using PVA, includes theproperty of PVA so slowly dissolve in liquid environments, such as inthe wound exudate, bodily fluids, etc., allowing for a slow release ofthe entrapped bacteriophages.

PVA can also be used for preparing simple and inexpensive spraydressing. Addition of PVA to bacteriophages in solution allows for theformation of a viscous solution that can be applied to a surface(wounds, plants, medical devices, implants, etc., forming an elasticfilm after evaporation of its water content, on the said surface. Again,PVA so slowly dissolve in liquid environments, such as in the woundexudate, bodily fluids, etc., allowing for a slow release of theentrapped bacteriophages.

Similarly, Poloxamer can be used for preparing such dressings. Poloxamerare modulable nonionic triblock copolymers: the central hydrophobicchain of polyoxypropylene and the two hydrophilic side chains ofpolyoxyethylene are customizable in terms of length. Poloxamer sol-gelproperties are of high interest: solutions of concentrated poloxamer areliquid at low temperatures and undergo sol-gel transition at highertemperatures. This process is reversible. For example Poloxamers 101,105, 108, 122, 123, 124, 181, 182, 183, 184, 185, 188, 212, 215, 217,231, 234, 235, 237, 238, 282, 284, 288, 331, 333, 334, 335, 338, 401,402, 403, and 407. can be used. In particular, thermoreversiblehydrogels are in situ forming gels that undergo sol-gel transition withan increase of temperature. Examples of such poloxamers are P188, P237,P338, and P407.

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non-restrictivedescription of preferred embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 , in an optical microscope image, illustrates Formulation 9,prepared according to example 1, with microcapsules containingStaphylococcus aureus phage J1P1

FIG. 2 , in an FEG-SEM image, illustrates amino-acid based microcapsulescontaining Staphylococcus phage BP39 prepared according to example 1;

FIG. 3 , in a bar chart ,illustrates the size distribution ofmicrospheres containing S. aureus phage BP39 prepared according toexample 1;

FIG. 4 , in a TEM image, illustrates bursting microtomied microspherescontaining phages prepared according to example 1;

FIG. 5 , in a TEM image, illustrates the portion of FIG. 4 found in thewhite dashed box; and

FIG. 6 , in a microscope image, illustrates formulation 9 of FIG. 1 42days after preparation (008 J1P1, 42 days old);

FIG. 7 , in an X-Y chart, illustrates microcapsule size distribution ina formulation similar to formulation 9; and

FIG. 8 , in an X-Y chart, illustrates microcapsules size distribution inthe formulation of Example 10.

DETAILED DESCRIPTION

The examples below use a polymer referred to as PEAU. This polymer is apoly (ester amide urea) comprising the following two blocks with randomdistribution thereof:

whereinthe ratio of l:m ranges from 0.05:0.95 to 0.95:0.05, l+m=1,R₁ is chosen from C₂-C₁₂ alkylenes optionally interrupted by at leastone oxygen, C₃-C₈ cycloalkylenes, C₃-C₁₀ cycloalkylalkylenes,

R₃ is C₂-C₁₂ alkylene,R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids so that the carbon to which R₂ or R₄ is attached has L orD chirality.

The more specific polymer referred to in the examples is the polymerwherein R₁ is —(CH₂)₆—, R₃ is —(CH₂)₈— and both R₂ and R₄ are the sidechain of L-leucine. These polymers are referred to hereinbelow inabreviated form as (8L6)_(l)-(1L6)_(m).

SUMMARY OF EXAMPLES 1 to 3

In example 1, the proposed method uses a high-speed homogenization of a13% PEAU solution in dichloromethane (DCM) with a 1% 67 kDa polyvinylalcohol (PVA) solution, dropped in a 2% PVA bacteriophages solution.After overnight evaporation of the solvent, the formulation in example 1is ready to use as is or for further formulations (see examples 2 and3). A portion of the bacteriophages remains in the external liquid, thatwill act as a short-time release (direct kill), and microcapsulescontain the remainder of the bacteriophages, and will allow a furtherlong-term release due to biodegradation. The formulation in example 2 isfabricated by adding 3% w/v of 67 kDa PVA to F9, the formulation ofExample 1 (final 5% w/v PVA). This formulation is to be used, forexample, as a film because high molecular weight PVA forms elastic filmafter evaporation of water, which is appropriated for sprays, amongother applications.

The 3^(rd) example is a gel formulation composed of the composition ofexample 1 supplemented by 25% w/v of Poloxamer P407. (This concentrationcan be modified at will to get the appropriate viscosity). PoloxamerP407 possess inverse thermosensitivity, which means it is liquid at lowtemperatures (2-4° C.) and will gel at high temperature (higher than 20°C.) so is useful for many applications, for example for the treatment of3^(rd)-degree burns.

Example 1: Preparation of Large Microcapsules Containing Bacteriophage

The following protocol was performed in this example:

1. Clean and sterilize all beakers, erlenmeyers, stoppers, funnels andvessels.2. Prepare the stock solution of Polyvinyl Alcohol (PVA) (1.0% w/v) inwater for the primary emulsion:a) put 100.0 mg (0.1 g) of PVA in a lab. flacon (20.0 mL capacity) or asmall beaker (max 50 mL)b) add 10.0 mL of waterc) put a magnetic stirring bar in the flacon/beakerd) place the flacon on a magnetic stirrere) switch the stirrer, select stirring rate 100-200 rpm (avoidsplashing)f) stir until complete dissolution of PVA (overnight)g) take the solution in a sterile syringe and pour through 0.22 umfilter in a sterile lab flacon under the laminar hoodh) place and store in a refrigerator (if not use in the same day).3. Prepare the PVA solution (2.0% w/v) in the liquid phages (or TMNbuffer depending on the task (TMN buffer can be used as a control)) forthe secondary emulsion:a) put 400.0 mg (0.4 g) of PVA in a sterile erlenmeyer, capacity max.200.0 mL under laminar hoodb) put a sterile magnetic stirring bar in the Erlenmeyer under laminarhoodc) add 20.0 mL of the liquid phage (or TMN buffer) under laminar hoodd) seal Erlenmeyer with a sterile foam under the laminar hoode) put the beaker on a magnetic stirrerf) switch the stirrer, select stirring rate 100-200 rpm (avoidsplashing)g) stir until complete dissolution of PVA (overnight)h) store in a fridge (if not used the same day)i) take the solution in a sterile syringe and pour through 0.22 umfilter in a sterile glass beaker (cap. 50 mL) under the laminar hoodj) cover with a sterile glass watch under the laminar hood4. Prepare the polymer solution (13.0% w/v) for the primary emulsion:a) put 1.04 g of the PEAU (8L6)_(0.6)-(1L6)_(0.40) in a small Erlenmeyerflask (max 50 mL)b) add 8.0 mL of DCM to the polymer in the flaskc) put a magnetic stirring bar in the flaskd) seal the flask with stopper wrapped in Teflon tapee) put the sealed small Erlenmeyer flask on the magnetic stirrerf) switch the magnetic stirrer on, select stirring rate 100-200 rpm(avoid splashing) and stir until complete dissolution of the polymerg) place it in a refrigerator (if not use the same day).5. Assemble the following set-up under the chemical hood: A smalldropping funnel with Teflon stopcock, capacity 20.0, max. 50.0 mL fixedto a lab stand, above a magnetic stirrer.6. Prepare the primary emulsion:a) open (remove stopper) the Erlenmeyer flask with the polymer solutionb) move the polymer solution (8 mL) to a sterile Lab flacon withstopper, capacity 20.0 mL, d=2.0-2.5 cm, under the chemical hoodc) take 0.8 mL from the PVA's stock solution (1%)d) quickly add this 0.8 mL to 8.0 mL the polymer solutione) put a dispersing element of a high speed homogenizer in the solutionf) switch the homogenizer, select the rate 10,000 rpmg) homogenize for 15 sec.h) remove the high speed homogenizeri) seal the flacon with the capj) immediately use for preparing the secondary emulsion7. Prepare the secondary emulsion (under chemical hood):a) remove the primary emulsion, from the flacon to the dropping funnelb) seal the dropping funnelc) put the glass beaker with PVA solution (2.0% w/v) in the liquidphages (or TMN buffer), on a magnetic stirrerd) it is preferable to fix the beaker to the lab stande) switch the magnetic stirrer and select stirring rate 400 rpm.f) remove the covering (watch glass) from the beakerg) add dropwise (ca. 1-2 drops per sec.) the primary emulsion from thedropping funnel to the beaker containing PVA's solution (2%) in theliquid phage (or TMN buffer)h) cover the beaker with the watch glass again (to prevent dust andbacterial invasion)i) stir at 400 rpm for 18 h until complete evaporation of DCMj) stop the stirrerk) go under the laminar hood to remove the covering (watch glass) fromthe beakerl) move the obtained MSs suspension to a sterile lab flacon with astopper of capacity max. 100 mL, seal the flacon with the stopper andstore the suspension in a refrigeratorm) use the obtained suspension for subsequent analysis

FIG. 1 is a microscopy image of a typical composition resulting fromthese manipulations. The microcapsules are clearly seen. They have asubstantially spherical shape with a hollow interior in which abacteriophage suspension is encapsulated. Scanning electron microscope(SEM) images were also acquired as follows. A drop of each wet sample ofmicrocapsules (obtained as per the method described above) was placed ona clean aluminum stub and dried under vacuum. The SEM (scanning electronmicroscope) observations were performed on a JEOL JSM-7600F equippedwith a field emission gun (FEG) operated at 1 kV. All the images wereacquired using the lower secondary electron detector (LEI). The imageanalysis was performed by measuring by hand the diameter of more than300 spheres on calibrated images using the Image) software(https://imagej.nih.gov/ij/).

An example of the resulting images is seen in FIG. 2 and the sizedistribution of the microcapsules (ie the polymer phase) is shown inFIG. 3 . The size distribution characteristics for the compositionincluding the BP39 phages were: minimum diameter: 11.15 μm; maximumdiameter: 304.77 μm; mean diameter: 60.72 μm and std deviation ofdiameter: 37.90 μm.

FIGS. 4 and 5 illustrate TEM images of the microcapsules (formulation 9,ie the formulation of example 1, also referred herein as F9), whichconfirms the presence of phages inside the microcapsules in F9. Morespecifically, detection of phages inside newly synthesized microcapsuleswas performed by fixing microcapsules by immersion with gluteraldehydeand dehydration with ethanol at 4° C. Tissue samples were embedded inEpon resin according to routine techniques and thin sections (100 nm) oftissues were processed by microtoming and mounted on Parlodion-carboncoated grids, counterstained with uranyl acetate, and examined underconventional transmission electron microscope at 80 kV. FIG. 5 is enenlargement of the dotted box of FIG. 4 .

Volatile organic compounds presence was evaluated as follows.Approximatively 500 mg of standard or samples (F9) were resolubilized in9 mL of water containing 7 g of ammonium sulphate and 200 μL of internalstandard, then heated for 1 hour at 120° C. Measurement was taken with aGC-MS from Agilent Technologies. 100 μL of gas was injected with a splitratio of 1:10. The temperature was set as following: 35° C. for 5 min,10° C./min to 90° C., 50° C./min to 250° C. Calibration curves wereprepared using DCM, cyclohexane and toluene. Internal standards usedwere cyclohexane-d12 and toluene-d18, cyclohexane-d12 being used tocorrect both the signal of DCM and cyclohexane. Results are summarizedin the following Table 1:

TABLE 1 Residual solvents concentration in F9 (ppm) DCM CyclohexaneToluene USP limits 600 3880 890 F9 (TMN) <60 <388 <89 F9 (BP39) <60 <388<89

This formulation thus does not contain any significant amounts ofresidual organic solvent and follows ISO requirements for medicaldevices. The values are below the detection of the method.

Stability was evaluated as follows. Samples were centrifugated slightlyand supernatant was pipetted. After titration, microcapsules wereresuspended. The formulations were stable for over 30 days at roomtemperature and 4° C., as illustrated in FIG. 6 illustrates a typical asseen through a microscope 42 days after preparation. The microscapsules'appearance is similar to the appearance just after preparationillustrated in FIG. 1 .

Phages titer inside microcapsules (MCs) were performed as follows.

Preliminary assay 1: 500 μL of F9 lot #011BP39 were taken andcentrifuged 3 min at 2000 RPM. Supernatant was removed (sample 1)—notethat the weight of the MCs was 0.05 g—and MCs were washed with 500 μL ofTMN, and centrifuged 3 min at 2000 RPM. Supernatant (sample 2) wasremoved and the wash was repeated (sample 2→7)—note that due the lossesduring the washes the weight of the MCs was then 0.04 g. Finally, 500 μLof DCM was added to break MCs. Approx. 17.5 μL of TMN solution wasrecovered from the MCs (sample D). Results are shown in Table 2.

TABLE 2 inside titration of phages in F9 lot #011BP39 in preliminaryassay 1 Sample 1 2 3 4 PFU/mL 7.50E+09 1.70E+09 1.90E+08 4.50E+07 Sample5 6 7 D PFU/mL 1.00E+07 4.60E+06 2.50E+06 1.30E+08

The results for D (dissolved microcapsules) is the sum of the result forthe last wash plus the phages liberated by the microcapsules. So the MCsliberated 1.28*10{circumflex over ( )}8 PFU/mL in the 17.5 μL2.23*10{circumflex over ( )}6 PFU, eg 5.58*10{circumflex over ( )}7PFU/g of MCs.

Preliminary assay 2: 1 mL of F9 lot #011BP39 were taken and centrifuged3 min at 2000 RPM. Supernatant was removed (sample 1)—note that theweight of the MCs was 0.1 g—and MCs were washed with 1 mL of TMN, andcentrifuged 2 min at 2000 RPM. Supernatant (sample 2) was removed andthe wash was repeated (sample 2→11). Finally, 1.5 mL of DCM and 100 μLof TMN were added to break MCs and dissolve phages. Less than the 100 μLof TMN solution were recovered from the MCs (sample D). In thisexperiment, it is clear that MCs broke during the washing process,regarding that the titers in the successive washed are not decreasing.Similarly to the previous assay an the amount of encapsulatedbacteriophages where of 4.8*10{circumflex over ( )}7 PFU/g of MCs.Results are found in Table 3.

TABLE 3 inside titration of phages in F9 lot #011BP39 in preliminaryassay 2 Sample 1 2 3 4 5 6 PFU/mL 7.40E+09 1.10E+09 1.70E+08 3.30E+072.80E+07 5.00E+06 Sample 7 8 9 10 11 D PFU/mL 3.50E+06 1.60E+06 1.00E+064.50E+05 4.10E+05 4.80E+07

Preliminary assay 3: 500 μL of F9 lot #011BP39 were taken andcentrifuged 3 min at 2000 RPM. Supernatant was removed (sample 1)—notethat the weight of the MCs was 0.05 g— and MCs were washed with 500 μLof TMN, and centrifuged 2 min at 2000 RPM. Supernatant (sample 2) wasremoved and the wash was repeated (sample 2→4 3). MCs were kept in2^(nd) wash (sample 3) and finally, 1 mL of DCM was added to break MCsand dissolve phages. TMN solution was recovered (sample 3*). Resultsfrom this assay are found in Table 4.

TABLE 4 Inside titration of phages in F9 #011_BP39 (preliminary test 3)Sample 1 2 3 3* PFU/mL 4.70E+09 5.00E+08 1.20E+08 2.80E+08

This method avoids the break of the MCs during the washes. Encapsulatedphage concentrations are of 1.6*10{circumflex over ( )}9 PFU/g of MCs.Moreover, a film of polymer forms at the interface between DCM and TMN.One drop of this interface was put on the titration plate (under theSaA29 writing), and shows a clear lysis. This means a lot of phages aretrapped in at the interface, that still contains MCs, which wasconfirmed through visual inspection.

Preliminary assay 4: 500 μL of F9 lot #011BP39 were taken andcentrifuged 3 min at 2000 RPM. Supernatant was removed (sample 1)—notethat the weight of the MCs was 0.04 g—and MCs were washed with 500 μL ofsterile water, and centrifuged 3 min at 2000 RPM. Supernatant (sample 2)was removed and the wash was repeated (sample 2→6). Last supernatant wasremoved (sample 6) and 1 mL of DCM was added to break MCs and dissolvephages. After 10 min, 100 μL of sample 6 was added to the mix, vortexedagain and centrifuged 5 min at 2000 RPM to separate phases. Sample F1was then taken in the aqueous phase. After another 5 min and vortexing,sample B was taken in a bubble of aqueous phase. Sample F3 was takenafter 30 min (after addition of DCM). The polymeric interphase was notpipetted because it was too small. This gives 3.58*10{circumflex over( )}7 PFU/g of MCs. Quantitative results are found in Table 5.

TABLE 5 Inside titration of phages in F9 #011_BP39 (preliminary test 4)Sample 6 F1 B F3 PFU/mL 3.60E+06 1.20E+07 1.70E+07 1.40E+07

Final titration: 500 μL of F9 were taken and centrifuged 3 min at 2000RPM. Supernatant was removed (sample 1) and MCs were washed with 500 μLof sterile water, and centrifuged 3 min at 2000 RPM. Supernatant (sample2) was removed and the wash was repeated (sample 2→6). Last supernatantwas removed (sample 6) and 1 mL of DCM was added to break MCs anddissolve phages. After 10 min, 100 μL of sample 6 was added to the mix,vortexed again and centrifuged 5 min at 2000 RPM to separate phases.Sample F was then taken in the aqueous phase. Quantitative results arefound in Table 6.

TABLE 6 Inside phages titers in formulations 9 Age of the sample atPhages titer Inside MCs Inside MCs the time before phages titer phagestiter F9 lot Date of of analysis encapsulation (PFU/g of (PFU/mL #production (days) Phages (PFU/mL) wet MCs) of F9) 003 6 Apr. 2016 63BP39  1.8 · 10¹¹ 2.17 · 10⁷  2.6 · 10⁶ 008 27 Apr. 2016 42 J1P1 6.5 ·10⁹ 4.91 · 10⁶ 5.89 · 10⁵ 009 27 Apr. 2016 42 J21P1 4.6 · 10⁸  6.5 · 10⁴ 7.8 · 10³ 010 27 Apr. 2016 42 J1P3 8.5 · 10⁸ 5.37 · 10⁵ 6.44 · 10⁴ 01127 Apr. 2016 42 BP39  3.6 · 10¹⁰ 3.58 · 10⁷ 3.58 · 10⁶ 013 16 May 201623 J1P1 6.5 · 10⁹ 1.52 · 10⁶ 1.52 · 10⁵ 014 16 May 2016 23 J21P1 4.6 ·10⁸ 1.49 · 10⁶ 8.92 · 10⁴ 015 16 May 2016 23 J1P3 6.5 · 10⁹ 1.23 · 10⁶1.23 · 10⁵ 016 16 May 2016 23 BP39  3.6 · 10¹⁰ 2.95 · 10⁶ 2.36 · 10⁵

Phages inside MCs and the phages trapped in the polymer or at thesurface are titrated by this method. The results seem to indicate thatphages are also stable inside the MCs, and that the higher the initialtiter is, the higher the encapsulated titer is. Prolonged storageexperiments showed that storage at 4° C. or room temperature is alsopossible for at least one year while preserving phage activity.

Example 2

This example aims at preparing a suspension with the bacteriophageloaded microcapsules (MCs) containing higher concentration (5% w/v) ofPVA (Formulation #10, also F10 hereinbelow). The following protocol wasperformed in this example:

1. Clean and sterilize all beakers, funnels, magnetic stirring bar, andvessels for storing the product.2. Prepare the phage loaded suspension according to the Protocol givenin example 1.3. When all DCM has been evaporated in Formulation 9, take either thebeaker with the glass watch or the flacons in which the MCs are storedunder the laminar hood.4. Put a sterile stirring bar in a sterile flacon with cap.5. Outside the laminar hood, weigh 600.0 mg (0.6 g) of solid PVA on asterile glass watch with a spatula previously plunged into alcohol7. Under laminar hood, transfer the powder in the sterile flacon with aspatula previously plunged into alcohol.8. Add 20 mL of resuspended (vortexed) Formulation 9 in the flacon witha sterile pipet of 20 mL.9. Put the sterile cap on the flacon.10. Put the flacon on the magnetic stirrer in the chemical hood for 24 huntil complete dissolution of the PVA.11. Stop the stirrer and store Formulation 10 at 4° C.12. Use the obtained Formulation 10 for subsequent tasks.

FIGS. 6 and 7 illustrate typical results preparations prepared accordingto the protocol of this example shortly after preparation (FIG. 6 ) andafter 23 days (FIG. 7 ).

Stability was quantified as follows. Samples were centrifuged slightlyand supernatant was pipetted. After titration, microcapsules wereresuspended. The MCs were stable and the phages remained active both at4° C. and at room temperature for at least one year.

Example 3

This example concerns preparation of the suspension with thebacteriophages loaded microcapsules (MCs) containing higherconcentration (25% w/v) of Poloxamer P 407 (Formulation #11, also F11below). Poloxamer P 407 is soluble in water at low temperatures (3-5°C.). The formulation will be gelled upon heating up to 20-25° C. orhigher temperatures (e.g. physiological temperature 36-37° C.). Thefollowing protocol was performed:

1. Clean and sterilize all beakers, funnels, magnetic stirring bar, andvessels for storing the product2. Prepare the phage loaded suspension according to the Protocol givenin example 1.3. When all DCM has been evaporated in Formulation 9, take either thebeaker with the glass watch or the flacons in which the MCs are storedunder the laminar hood.4. Put a sterile stirring bar in a sterile flacon with cap.5. Outside the laminar hood, weigh 5.0 g of solid Poloxamer P 407 on asterile glass watch with a spatula previously plunged into alcohol.6. Under laminar hood, transfer the powder in the sterile flacon with aspatula previously plunged into alcohol.7. Add 20 mL of resuspended (vortexed) Formulation 9 in the flacon witha sterile pipet of 20 mL.8. Put the sterile cap on the flacon.9. Put the flacon on the magnetic stirrer in the fridge for 24 h untilcomplete dissolution of the Poloxamer.10. Stop the stirrer and store Formulation 11 at 4° C.11. Use the obtained Formulation-11 for subsequent tasks.

This preparation's stability was assessed as above. It was stable for upto more than a one year at 4° C. Since this preparation becomes a gel athigher temperatures, stability at room temperature was not assessed.

Microencapsulated formulations of examples 1, 2 and 3 (liquid spray,spray-patch, and spray-gel) containing cocktails of phages are stableand maintain their activity at 4° C. and room temperature. Stability ofthe formulation and activity of the encapsulated phages are still stableat room temperature, we are at time point 220 days for the liquid spray,284 days for the Spray-Patch, and 288 days for the spray-gel. Moredetailed results are presented in Table 7 below

TABLE 7 Stability of the preparations of examples 1 to 3 Stability andactivity of encapsulated Individual/ formulations Formulation Cocktail4° C. 22° C. 37° C. Liquid spray Individual Ongoing, 113-over 277  5-30days (Formulation 9 of over 1 year days example 1) Cocktail Ongoing,Ongoing, 220   20 days 220 days days Spray-patch Individual Ongoing,84-250 days 15-60 days (Formulation 10, over 1 year 5% PVA of CocktailOngoing, Ongoing, 284   15 days example 2) 284 days days Spray-gelIndividual Ongoing, Over 230 days 15-58 days (Formulation 11, over 1year Poloxamer of Cocktail Ongoing, Ongoing, 288   15 days example 3)288 days days

Example 4: Rough Polymeric Microcapsules

In another embodiment, the bacteriophages are adsorbed by inorganicsalts—calcium carbonate (CaCO₃) and calcium phosphate (Ca₃(PO₄)₂), priorto formation of microcapsules.

We have repeatedly studied the adsorption of bacteriophages by the waterinsoluble salts of calcium—CaCO₃ and Ca₃(PO₄)₂. In these experiment newphages preparations TG-1 and TG-2 were used. The data listed in Table 8confirm that the bacteriophages are adsorbed by these salts.

TABLE 8 Insoluble calcium salts containing adsorbed bacteriophages PFU *Phages Phages Bacteriophages TG1 CaCO₃* TG2 Ca₃(PO₄)₂* 1 Staphylococcus10⁸ 10⁵ 10⁹ 10⁷ 2 E. coli 10⁹ 10⁶ 10⁹ 10⁸ 3 Streptococcus 10⁸ 10⁶ 10⁸10⁷ 4 Ps. aeruginosa 10⁸ 10⁶ 10⁹ 10⁷ 5 Proteus 10⁸ 10⁶ 10⁸ 10⁸ * PFU ofthe bacteriophages desorbed from the salts.

These salts can be great products for the application in agriculture totreat plants. Calcium carbonate is used for decreasing acidity of thesoil, and calcium carbonate is used as a fertilizer. Hence, the saltswith absorbed bacteriophages can fulfill (at least) double function—tokill parasitic bacteria and to refine the soil. The salts are of a highinterest for the application in livestock as well, e.g. to treat animalsfrom pathogenic form of E. coli (carbonate salt will protect thebacteriophages from the inactivation by the acidic medium of stomach).The salts are also highly promising for medical applications as well inbone surgery, dentistry, etc.

These salts can be applied as either dry powders or in combination withPVA and Poloxamer. With these polymers (industrial products) thepulverizable composite can be obtained. The vehicles such as PVA andPoloxamer will allow to better fixing the salts' particles at varioussurfaces.

In some embodiments, the salts are incorporated in grindable polymericmicroparticles forming the microcapsules. For example the polymer is alow molecular weight (LMW) polymer based on 1L6. The protocol forpreparing bacteriophage loaded rough polymeric microparticles is givenbelow.

In an exemplary procedure, 2.0 g of LMW-1L6 was dissolved in 20.0 mL ofDCM at room temperature and 2.0 g of dry calcium carbonate (CaCO₃) withmean particle size 6.2 μm was used for this purpose) containing absorbedbacteriophages and was thoroughly homogenized for 15 min at 22,000 rpmusing a high speed-homogenizer, and cast onto a Teflon plate, DCM wasevaporated at room temperature and atmospheric pressure until dried, andthe obtained brittle film was subsequently dried under vacuum at 30° C.for 20 hours. The film was removed from the Teflon backing, grindedmechanically in a porcelain pounder and sieved through 90 μm mesh-sizesieve. The results of particles size and size distribution determinationfollowing grinding and sieving through 90 μm mesh-size sieve are givenin Table 9. The particles mean size right after grinding was 65.28 μmand upper limit size was 175.47 μm which is acceptable for preparingsuspension used for purpose of spraying surfaces, such as wounds, usinga spraying apparatus with the a nozzle diameter of 250-300 μm. Mean sizeof the particles decreased to 35.49 μm, with an upper limit size was89.03 μm after sieving through 90 μm mesh. The biocomposites weresubsequently tested for phage activity.

TABLE 9 Particle size and size distribution after grinding biocompositeon the basis of LMW-1L6. Lower limit Upper limit Mean size size sizeMeasurement (μm) ± SD (μm) ± SD (μm) ± SD Right after grinding 65.28 ±6.393 10.21 ± 0.584 175.47 After sieving through 35.49 ± 4.532  8.86 ±0.474 89.03 ± 1.432 90 μm mesh

To further optimize the process, a high-speed homogenizer was used todisintegrate the solid particles. The initial suspensions was preparedby mixing 2 g of CaCO₃, with mean particle size 6.2 μm, with 20 mL ofwater. Homogenization of the suspension for a period 15 sec and 5 mindid not show a significant effect on particle size. Increasing thehomogenization time to 15 min showed tangible effect where particle sizedecreased to 3.27 nm. Further increasing the homogenization time to 30min gave only a slight diminishing particles size (3.10 nm). Moredetails are provided in Table 10.

TABLE 10 Particles size and size distribution of CaCO₃ powder before andafter the treatment with a high speed homogenizer. 2 g of CaCO₃ in 20 mLof water, at 22,000 rpm. Particles Particles lower Particles upper Timeof the mean size limit size limit size treatment, min. (μm) ± SD (μm) ±SD (μm) ± SD Initial powder 6.20 ± 0.013 1.99 ± 0.005 13.03 ± 0.057 (aspurchased) 0.25 5.59 ± 0.068 1.50 ± 0.015 12.23 ± 0.087 5 5.23 ± 0.0431.54 ± 0.021 11.02 ± 0.198 15 3.27 ± 0.027 0.99 ± 0.016  6.79 ± 0.056 303.10 ± 0.026 0.95 ± 0.040  6.50 ± 0.114

This experiment showed that the high speed homogenizer can be usedinstead of mechanical grinding in a porcelain pounder for preparingbacteriophage loaded rough polymeric microparticles.

Preparation of Phage Loaded Microparticles

The preparation of rough polymeric microsparticles (MPs) is a simpletechnology allowing for the encapsulation of bacteriophages and otherphage products. In this embodiment, an insoluble inorganic salt (e.g.CaCO₃) containing adsorbed bacteriophages is added to a solution ofLMW-1L6 in chloroform or DCM (or another organic solvent), thesuspension is thoroughly homogenized, cast onto a hydrophobic surface,the solvent is evaporated, the fabricated brittle film is grinded andthe obtained powder is sieved through 90 μm mesh-size sieve.

In addition to this technology we elaborated another simple technologywhich is based on the use of the insoluble inorganic salt (e.g. CaCO₃)with absorbed bacteriophages. According to the new technology theinorganic particles, “bearing” surface immobilized bacteriophages, areencapsulated (“wrapped”) in the biodegradable polymeric capsule byoil-in-water (O/W)-solvent evaporation method.

In another embodiment, 0.1 g of the biodegradable polymer 8L6 wasdissolved in 5 mL of DCM, 0.5 mL of water was added to this solution andhomogenized at 10,000 rpm for 1 minute using a high-speed homogenizer,thus obtaining a polymeric emulsion. In a separate vessel 2 g of CaCO₃with adsorbed phages was added to 50 mL of 2.5% PVA water solution andstirred for 20 min using a magnetic stirrer. The previously preparedpolymeric suspension was added to the PVA solution in a drop-wisemanner, while mixing at 22,000 rpm. After the emulsion was completelyadded, mixing was sustained for another 3 min. Afterwards, the obtainedsuspension was placed on a magnetic stirrer and stirred (450-500 rpm)for 4 h until the complete evaporation of DCM. Table 11 shows the sizeand size distributions of the obtained microcapsules. Resuspension ofthe microparticles following gravimetrical precipitation andfreeze-drying did not change the properties of the microcapsules.

TABLE 11 The characteristics of MPs obtained by encapsulation ofinsoluble “bacteriophage bearing” inorganic particles (CaCO₃) inbiodegradable amino acid based poly(ester amide) 8L6. Lower Upper Thesalt with adsorbed Mean limit limit Phages, subjected to diameterdiameter diameter # encapsulation with 8L6 (μm) ± SD (μm) ± SD (μm) ± SD1 CaCO₃ (as obtained) 5.01 ± 0.01 1.67 ± 0.01 11.12 ± 0.08 2 CaCO₃,re-suspended after 5.55 ± 0.04 1.76 ± 0.01 11.82 ± 0.06self-precipitation (gravimetrically) 3 CaCO₃, re-suspended after 5.02 ±0.05 1.77 ± 0.01 10.06 ± 0.25 freeze-drying of the initial suspension(sample #1)Standard protocol for determining activity of bacteriophages (in termsof plaque assay—PFU) for obtained solid preparations.

Determination of bacteriophage activity was obtained dissolving one gramof the solid in 4 mL of buffer at room temperature. The flask is placedon a shaker 30 min. and standard microbiological plaque assays areperformed.

Activity of the CaCO₃ and Ca₃(PO₄)₂ with absorbed bacteriophagessubsequently encapsulated in biodegradable poly(ester amide) 8L6 isgiven in Table 12.

TABLE 12 Insoluble calcium salts containing adsorbed bacteriophagesencapsulated in the biodegradable poly(ester amide) 8L6. PFU of the PFUof the bacteriophages bacteriophages desorbed desorbed from the from theencapsulated encapsulated Initial phage in poly Initial phage in polyconcentrations (ester amide) concentrations (ester amide) TG1 8L6 CaCO₃in TG2 8L6 Ca₃(PO₄)₂ Bacteriophage solution salts solution salts 1Staphylococcus 10⁸ 10⁴ 10⁹ 10⁵ 2 E. coli 10⁹ 10⁶ 10⁹ 10⁷ 3 Streptococcus10⁸ 10⁵ 10⁸ 10⁵ 4 Ps. aeruginosa 10⁸ 10⁵ 10⁹ 10⁶ 5 Proteus 10⁸ 10⁴ 10⁸10⁴

Effect of Particle Size and Size Distribution

Inorganic salts such as calcium carbonate (CaCO₃) and calciumtri-phosphate (Ca₃(PO₄)₂ are used for preparing powdery preparation withadsorbed bacteriophages (see below). Those are vastly available and verycheap products. Purchasable calcium carbonate consists of microparticlesof rather small size—mean size ca. 6 μm, and upper size 13 μm—both quiteacceptable for preparing spray preparations for both medical andagricultural applications. We have found that after the treatment withhigh-speed homogenizer for 15 min particle size (both mean and uppersizes) could be diminished ca. twice (Table 13, sample #4).

TABLE 13 Particles size and size distribution of CaCO₃ powder before andafter the treatment with HS-homogenizer at 22,000 rpm. 2 g of CaCO₃ in20 mL of water. Particles Particles Particles mean lower limit upperlimit Time of the diameter diameter diameter Particles # treatment, min.(μm) ± SD (μm) ± SD (μm) ± SD R* distribution 1 Initial powder 6.20 ±0.013 1.99 ± 0.005 13.03 ± 0.057 1.78 wide (as purchased) 2 0.25 5.59 ±0.068 1.50 ± 0.015 12.23 ± 0.087 1.92 wide 3 5 5.23 ± 0.043 1.54 ± 0.02111.02 ± 0.198 1.81 wide 4 15 3.27 ± 0.027 0.99 ± 0.016  6.79 ± 0.0561.77 wide 5 30 3.10 ± 0.026 0.95 ± 0.040  6.50 ± 0.114 1.79 wide *R =(Upper limit diameter − Lower limit diameter)/Mean diameter; R >1.5—wide particle distribution, R < 1.5—narrow particle distribution.

The same was observed with calcium tri-phosphate. Commercial powderscontained larger particles with mean size ca. 31 μm, and upper size ca.59 μm. High speed homogenization for 3 min at lower speed 10,000 rpmreduced the size of the microparticles. The subsequent treatment up to15 min led to a reduction of size with a mean diameter of 9.75 μm (mean)and an upper limit of 26.31 μm. More detailed results are found in Table14.

TABLE 14 Particles size and size distribution of Ca₃(PO₄)₂ powder beforeand after the treatment in water with HS-homogenizer at 10,000 rpm. 2 gof Ca₃(PO₄)₂ in 20 mL of water. Mean Lower limit Upper limit Time of thediameter diameter diameter # treatment, min. (μm) ± SD (μm) ± SD (μm) ±SD 1 Initial powder 30.97 ± 0.226  9.52 + 0.051 59.29 ± 0.691 (aspurchased) 2  3 min 16.38 ± 0.105  4.74 ± 0.043 37.41 ± 0.287 3 15 min 9.75 ± 0.355  3.85 ± 0.029 26.31 ± 0.630 4 PVA 55.68 ± 1.929 30.78 ±0.021 85.01 ± 0.198 3 min with 2.5% PVa (foam) 4 #3 with subsequent22.91 ± 0.55  6.05 ± 4.83 50.54 ± 0.32 mixing on a magnetic stirrer at450 rpm for 3 h. A form partly disappeared

As was mentioned previously, water insoluble inorganic salts such ascalcium carbonate and calcium phosphate strongly adsorb thebacteriophages from the solution. Our previous date were confirmed whenworking with cocktails of bacteriophages—TG-1 and TG-2.

The particles mean sizes of the commercial salts are of micrometer scaleand can be directly used for preparing micro-emulsions. However, we haveshown that, the particles sizes and size distribution can be narrowedafter the treatment with a high-speed homogenizer. Owing to particlessize the salts can be used as pulverizable solids (powders). Such formcan be useful for dressing exudative wounds. Moreover, they can beprepared also as pulverizable liquids (i.e. pulverizable suspension) bymixing with PVA's or Poloxamer's solutions which solutions can beprepared on the basis of either liquid bacteriophages or saline solutionas described hereinabove.

Example 5: Different Formulations for Different Applications

Non-limiting examples of applications for formulations containingencapsulated bacteriophages are provided below.

Formulation #1: Pulverizable Liquid

A simple and inexpensive pulverizable formulation for adheringbacteriophages on surfaces can be obtained by dissolvinghigh-molecular-weight poly(vinyl alcohol) (PVA, MW 84,000-89,000 Da) ina bacteriophage solution at room temperature. For example, theconcentration of the PVA is 2.5-5.0% (w/v). Spraying such preparationson surfaces (wound surface, plant surface, etc.) will allow theformation of an elastic PVA film impregnated with bacteriophages, afterthe evaporation of water. The PVA film will release phages as it canslowly dissolve in liquid found in the environment where it issprayed/applied (bodily fluids, plants' irrigation system). This allowsfor a slow (prolonged) release of bacteriophages from the coating. Theformulation can be loaded with other medicines (antibiotics, painkillers, hemostatics, etc.) along with bacteriophages.

An example of this formulation was prepared by dissolution of PVA (2.5%w/v; PVA content can be increased up to 5% or higher) in the liquidPhages TG-2 at room temperature and the obtained solution was kept in arefrigerator for three weeks. After three weeks PFUs of the phages of F1were determined. The PFUs of the phages are retained at the initiallevel, in other words PVA did not inactivate Phages during three months.

Formulation #2: Pulverizable Liquid

Similarly, dissolving the Poloxamer 407 (P-407, MW 9,840-14,600 Da) inliquid bacteriophage solution at a temperature 5° C. An non-limitingexample of P-407 concentration is 20% (w/v). Such preparation when beingsprayed on warm surfaces (wound surface, plant surface, etc.) forms gellike coating impregnated with bacteriophages (in case of P-407 there isno need in water evaporation—the gelation takes place upon warming theformulation). The P-407 gel will release phages as it can slowlydissolve in liquid found in the environment where it is sprayed/applied(bodily fluids, plants' irrigation system). This allows for a slow(prolonged) release of bacteriophages. This formulation, due to the gelproperties could be suitable for wounds, particularly for burns.

An example of the formulation #2 was prepared by dissolution ofPoloxamer 407 (20% w/v) in the liquid preparation of phages TG-2 at roomtemperature and the obtained solution was kept in a refrigerator forthree months. After three weeks PFUs of the phage of the F2 weredetermined. The concentration of the phages remained unchanged and equalto the initial level during three months. The P-407 gel can be loadedwith various medicines in combination with bacteriophages or phageproducts, and can be used for superficial wound healing purposes.

Formulation #3: Pulverizable Powder

Those are water insoluble salts like calcium and magnesium carbonates,calcium and magnesium phosphates (as well as their mixtures) containingsurface adsorbed bacteriophages, in general labeled as St/BP. Otherwater insoluble salts of other metals such a barium, strontium, etc. canbe used as well.

Formulation #4: Pulverizable Suspension

As noted above the St/BP can be used as pulverizable powders. However,in case it is desirable to fix the St/BP particles at surfaces (woundsurface, plant surface, etc.), one can use formulations as suspensionsof the St/BP in a solution of PVA in saline or liquid bacteriophage(2.5-5% (w/v) of PVA). Such formulation when being sprayed on thesurface, forms an elastic film of PVA which is impregnated with theSt/BP particles. The film of PVA will release phages as it can slowlydissolve in liquid found in the environment where it is sprayed/applied(bodily fluids, plants' irrigation system). This allows for a slow(prolonged) release of the St/BP particles. The formulation can beloaded with other medicines (antibiotics, pain killers, hemostatics,etc.) along with St/BPs.

Formulation #5: Pulverizable Suspension

To fix the St/BP particles at surfaces (wound surface, plant surface,etc.) the P-407 (or other poloxamers) can be used instead of the PVA.For example, formulations can be made as suspensions of the St/BP in asolution of the P-407 in saline or liquid bacteriophage solution(20%-25% (w/v) of the P-407 prepared at 5° C.). Such formulations whenbeing sprayed on warm surfaces (wound surface, plant surface, etc.) forma gel-like coating impregnated with the St/BP particles (P-407 is in aliquid form at low temperatures but gels upon warming the formulation tobodily temperatures). P-407 gel will release phages as it can slowlydissolve in liquid found in the environment where it is sprayed/applied(bodily fluids, plants' irrigation system). This allows for a slow(prolonged) release of the particles with the adsorbed bacteriophages.The formulation can be loaded with other medicines (antibiotics, painkillers, hemostatics, etc.) along with St/BP.

Formulation #6: Pulverizable Powder

This product represents the St/BPs encapsulated in an amino acid basedbiodegradable polymer covered by the present patent application. Theproduct is labeled as St/BP/En.

An advantage of encapsulating St/BPs encapsulated in an amino acid is toslow down the bacteriophage desorption from the salts, i.e. to make thedesorption more controllable. Besides, the existence of the amino acidbased biodegradable polymer is highly desirable when the product is usedfor wound healing processes as the polymer stimulates the wound healing,presumably through activating macrophages.

Formulation #7: Pulverizable Suspension

As noted above the St/BP/En can be used as pulverizable powders.However, if it is desirable to fix the St/BP/En particles on surfaces(wound surface, plant surface, etc.) formulations in the form ofsuspensions of the St/BP/En in a solution of PVA in saline or liquidbacteriophage (2.5-5% (w/v) of the PVA, for example) could be used. Suchformulations when being sprayed on the surface form an elastic film ofPVA which is impregnated with the St/BP/En particles. The film of PVAwill release phages as it can slowly dissolve in liquid found in theenvironment where it is sprayed/applied (bodily fluids, plants'irrigation system). This allows for a slow (prolonged) release of theSt/BP/En particles. The formulation can be loaded with other medicines(antibiotics, pain killers, hemostatics, etc.) along with St/BP/En.

Formulation #8: Pulverizable Suspension

To fix the St/BP/En particles on surfaces (wound surface, plant surface,etc.) the P-407 can be used instead of the PVA. Such formulationsinclude for example suspensions of the St/BP/En in a solution of theP-407 in saline or liquid bacteriophage solution (20% (w/v) of the P-407prepared at 5° C., non-limitingly). Such formulations, when sprayed onwarm surfaces (wound surface, plant surface, etc.), form gel-likecoating impregnated with the St/BP/En particles (in case of P-407 thereis no need in water evaporation—the gelation takes place upon warmingthe formulation). The P-407 gel will release phages as it can slowlydissolve in liquid found in the environment where it is applied (bodilyfluids, plants' irrigation system). This allows for a slow (prolonged)release of the St/BP/En particles. The formulation can be loaded withother medicines (antibiotics, pain killers, hemostatics, etc.) alongwith St/BP/En.

Formulation #9: Pulverizable Suspension

According to the W/O/W double emulsion method we elaborated, thesuspension of microcapsules, also referred to as microspheres, with thebacteriophages in its interior (MS/BPI) suspended in a solutioncontaining bacteriophages is obtained. This suspension can be useddirectly, without separating the MS/BPI from the surrounding liquidcontaining bacteriophages. The phages both entrapped in the MS/BPI andin the liquid phase (i.e. out of microspheres—in bacteriophage solution)will actively participate in the wound healing processes on two fronts:direct kill (provided by the free phages in the surrounding liquid) andprolonged release (provided by phages entrapped in the polymericmicrocapsules). Such formulations (free of additive such as vehicles—PVAor P-407, see below) could be useful as a food additive. The formulationcan be loaded with other medicines (antibiotics, pain killers,hemostatics, etc.) along with MS/BPI.

Formulation #10: Pulverizable Suspension

As noted above, the suspension of MS/BPI in the serial liquidbacteriophage can be used as pulverizable formulation. However, in caseif there is a need to fix the MS/BPI particles at surfaces (woundsurface, plant surface, etc.), it is possible to use formulations assuspensions of the MS/BPI in a solution of PVA. The formulations can beloaded with other medicines (antibiotics, pain killers, hemostatics,etc.) along with free bacteriophages and MS/BPI.

Examples of the Formulation #10 was prepared as follows: awater-in-oil-in-water (w/o/w) double emulsion-solvent evaporation methodwas employed to fabricate the biodegradable MSs with bacteriophages inthe interior microcapsule space (i.e phages encapsulated in thebiodegradable MSs). Briefly, the primary emulsion was prepared by 4.0 mL1% w/v aqueous Kolliphor P188 (Surfactant 1) into 40.0 mL 5% w/v 8L6 inDCM and by homogenizing for 15 s at 10,000 rpm using a homogenizer (Highshear strength disperse homogenizing emulsification machine C25). Thisprimary emulsion was added to 100 mL of Phages TG-2, containing 2.5 or5.0% of dissolved PVA with Mw=84,000-89,000 (Surfactant 2), and washomogenized for 3 min at 10,000 rpm. This w/o/w emulsion was immediatelystirred using magnetic stirrer for 18 hours to evaporate organicsolvent. The obtained suspension free of DCM was cast onto hydrophobizedPetri-dishes and dried in a vacuum-drier at r.t. for 48 h over anhydrousNa₂SO₄ to remove water. The dried films containing both free phages andphages encapsulated in the biodegradable MSs were subjected to plaqueassays to quantify phage concentrations. Phage activity was maintainedafter storage for at least one week.

In another example, a water-in-oil-in-water (w/o/w) doubleemulsion/solvent evaporation method was employed to fabricatebacteriophage loaded microspheres. Briefly, the primary emulsion wasprepared by mixing 8 mL of 1% w/v water solution of Kolliphor P188 and80 mL 5% w/v solution of poly(ester amide) 8L6 in dichloromethane (DCM)and by homogenizing for 15 s at 10,000 rpm. This primary emulsion wasadded to 200 mL bacteriophage concentrate (TG-2), containing 1% oflow-molecular-weight polyvinyl alcohol (PVA, MW 13-23 KDa) andhomogenized for 3 min at 10,000 rpm. This w/o/w emulsion was immediatelymoved to a magnetic stirrer and stirred for 24 hours to evaporate theorganic solvent. Then prepared suspension was dialyzed against salinesolution for 2 weeks and lyophilized over 48 h using TOPT-10C Freezingdryer, TOPTION, China (vacuum of 1 Pa, condenser—59° C., samplecompartment +17° C.) and stored in a refrigerator.

Lytic activities of bacteriophages were measured after a month storageat 4° C. Bacteriophages were desorbed from the dried MSs according tothe following protocol. 1.0 g of the freeze-dried MSs are placed in aflask and 4.0 mL of PBS at room temperature. The flask is placed on ashaker and shaken for 3 and 24 hours. After the shaking is stopped, theflask is kept for 5 min without agitation and aliquot is taken off fromthe supernatant for determining PFU.

Bacteriophages encapsulated in the MSs are active after one monthstorage in a refrigerator. Observed higher (by 10¹-10²) PFUs of thephages after 24 h shaking as compared with 3 h desorption could beascribed to increasing the phages concentration in the liquid phase(saline solution) owing to the release of the phages from the MSs.

Formulation #11: Pulverizable Suspension

To fix the MS/BPI particles on surfaces (wound surface, plant surface,etc.) the P-407 can be used instead of the PVA, for example 20% (w/v) ofthe P-407 prepared at 5° C. Such formulation when being sprayed on warmsurfaces (wound surface, plant surface, etc.) forms a gel-like coatingimpregnated with the MS/BPI particles. The P-407 gel gel will releasephages as it can slowly dissolve in liquid found in the environmentwhere it is applied (bodily fluids, plants' irrigation system). Thisallows for a slow (prolonged) release of the MS/BPI particles. Theformulations can be loaded with other medicines (antibiotics, painkillers, hemostatics, etc.) along with free bacteriophages and MS/BPI.

Example 6: Larger Scale Manufacturing and Stability

This example aims at preparing a suspension with the bacteriophageloaded microcapsules (MCs) containing 1% (w/v) of PVA, a variant ofFormulation #10 (F10′ hereinbelow). The following protocol was performedin this example:

-   -   Clean glassware and equipment with 2% CIP100 (in type I water)        for 2 hours at room temperature. Rinse glassware and equipment        thoroughly with type I water.    -   Clean glassware and equipment with 2% CIP200 (in type I water)        for 2 hours at room temperature. Rinse glassware and equipment        thoroughly with type I water.    -   Depyrogenize glassware and equipment with 0.5N NaOH (in type I        water) for 2 hours at room temperature. Rinse glassware and        equipment thoroughly with type I water until neutral pH.        Sterilize glassware and equipment with saturated vacuum at        121° C. and 15 psi for at least 15 minutes.    -   Assemble Rotor Stator Homogenizer over a 2 L pyrogen-free and        sterile beaker under a BSC. Assemble Overhead Mixer equipped        with a 4 in stainless steel windmill blade over a 2 L        pyrogen-free and sterile beaker under the same BSC.    -   PEAU 11% (w/v) in DCM: Dissolve 104.0 g of PEAU in 945 mL of DCM        for a final concentration of 11% (w/v) in borosilicate glass jar        (2 L) with a depyrogenized and sterile stirring bar. Stir for 2        to 3 hours at convenient speed until complete dissolution. A        total of 9.45 L of 11% (w/v) PEAU in DCM is prepared.    -   PVA 11% (w/v) solution: Dissolve 242.0 g of PVA in 2.2 L of 10%        TMN buffer (5 mM of Tris-HCl at pH 7.4, 1 mM of MgSO₄ and 10 mM        of NaCl in water for injection) with continuous agitation for        16-18hours at 50° C. Sterile filter the 11% (w/v) PVA solution        with 0.22 μm filter.    -   Cocktail-SPK at 5.9E10PFU/mL+1.0% (w/v) PVA for primary        emulsion: Dissolve 8.01 g of PVA in 800 mL of Cocktail-SPK        (including bacteriophages specific to P. aeruginosa, S. aureus,        and K. pneumoniae) at 5.9E10PFU/mL with continuous for 16-18        hours at 4° C. Sterile filter the solution with 0.22 μm filter        under a biological safety cabinet.    -   Secondary emulsion in 50 L glass reactor: Pour 20 L of        Cocktail-SPK at 5.9E09PFU/mL in 50 L glass reactor and add 2.2 L        of PVA 11% (w/v) solution. Stir the second emulsion in reactor        kept under positive pressure (using sterile-filtered nitrogen at        3.5 L/min).    -   Primary emulsion: Pour 1 L of PEAU 11% (w/v) in DCM and 80 mL of        Cocktail-SPK at 5.9E10PFU/mL+1.0% (w/v) PVA in the 2 L beaker        (step 5). Homogenize primary emulsion for 60 seconds at 20,000        RPM. Transfer the primary emulsion to the 2 L beaker (step 6)        and stir at 100-300 RPM to maintain emulsion homogeneity.        Another batch of primary emulsion is prepared when only 400 mL        is left in the beaker (step 6).    -   Formulation preparation: Transfer primary emulsion from 2 L        beaker (step 6) to the 50 L reactor containing the second        emulsion stirred at 250 RPM. Transfer is performed using a        peristaltic pump and a feed tube with extremity placed below        secondary emulsion surface.    -   DCM evaporation: Maintain reactor agitation at 200 to 350 RPM        for 18h (at room temperature) with a nitrogen feed at a rate of        3 L/min through a 1 in tube. Continue DCM evaporation for 48 h        at 34° C. Confirm DCM removal using GC-MS analysis.    -   Formulation transfer: Transfer formulation to pyrogen-free and        sterile 20 L polypropylene carboy.

Then, phages titer inside microcapsules (MCs) were performed as follows.

-   -   One milliliter (in triplicate) of F10′ formulation is        centrifuged for 5 minutes at 2000 RPM and 4° C. Supernatant is        removed and stored at 4° C. for titration of bacteriophages        present in free suspension.    -   Microcapsules pellet (350-400 μL) is then washed 3 times with 1        mL of 10% TMN buffer with a centrifugation step (conditions        indicated above) after each wash cycle.    -   After removal of the last washing buffer, microcapsules pellet        is resuspended in 100 μL of chloroform and vortexed at maximal        speed for 20 s.    -   One milliliter of 10% TMN buffer is added to each tube and        mixture is vortexed at maximal speed for 15 s.    -   Tubes are centrifuged at the same conditions indicated above and        the aqueous phases containing phages extracted from        microcapsules are cautiously collected in clean tubes.        Phages present in free suspension and extracted from        microcapsules are titered on corresponding production host and        pathogenic bacteria. Results are shown in table 15.

TABLE 15 Titer of microencapsulated phages in F10′ in PFU/mL Initialcocktail Initial for free cocktail to be Microencapsulated suspensionmicroencapsulated Free suspension phages S. aureus 4.50E09 4.35E101.89E09 ± 3.53E08 9.40E07 ± 5.03E07 P. aeruginosa 2.70E08 2.55E092.93E08 ± 1.53E07 8.05E07 ± 1.95E07 K. pneumoniae 3.80E08 2.80E092.90E08 ± 7.80E07 4.04E07 ± 8.81E05 Complete 5.05E09 4.86E10 2.48E09 ±2.87E08 2.15E08 ± 6.40E07 cocktailStability of formulation F10′ was investigated as follows. FormulationF10′ was stored in 10 mL glass vials at 4° C. Sampling was performedaccording to USP<1049>; i.e. At 0, 2, 4 weeks, 3 and 6 months. At eachtimepoint, microencapsulated bacteriophages were extracted usingchloroform method as hereinabove in the present example andbacteriophages present in free suspension and extracted frommicrocapsules were titered on corresponding production host andpathogenic bacteria. Detailed results are presented in the table below.

TABLE 16 Stability of lot F10′ stored in glass vials at 4° C. (phagetitre in PFU/mL) Microencapsulated Microencapsulated Free suspensionphages Free suspension phages Timepoint 0 2 weeks S. aureus 1.89E09 ±3.53E08 9.40E07 ± 5.03E07 5.73E08 ± 5.87E07 7.26E07 ± 3.16E07 P.aeruginosa 2.93E08 ± 1.53E07 8.05E07 ± 1.95E07 2.70E08 ± 7.94E07 7.00E07± 3.50E06 K. pneumoniae 2.90E08 ± 7.80E07 4.04E07 ± 8.81E05 1.52E08 ±3.71E07 4.94E07 ± 2.90E07 Complete 2.48E09 ± 2.87E08 2.15E08 ± 6.40E079.95E08 ± 4.70E07 1.92E08 ± 2.90E07 cocktail Timepoint 4 weeks 12 weeksS. aureus 1.51E09 ± 3.00E08 1.10E08 ± 1.65E07 1.50E09 ± 1.49E09 1.22E08± 1.17E07 P. aeruginosa 3.50E08 ± 7.00E07 6.42E07 ± 1.33E07 3.33E08 ±4.51E07 6.24E07 ± 3.65E07 K. pneumoniae 2.46E08 ± 3.16E07 5.82E07 ±1.99E06 1.99E08 ± 1.42E07 1.40E08 ± 1.84E08 Complete 2.11E09 ± 3.03E082.32E08 ± 3.09E07 2.03E09 ± 1.52E09 3.25E08 ± 2.12E08 cocktail Timepoint24 weeks S. aureus 2.79E08 ± 9.17E07 6.57E07 ± 8.80E06 P. aeruginosa1.70E08 ± 2.65E07 5.37E07 ± 5.35E06 K. pneumoniae 1.72E08 ± 1.63E074.52E07 ± 8.17E06 Complete 6.21E08 ± 7.31E07 1.65E08 ± 8.00E06 cocktail

Then, formulation F10′ was diluted 5 folds in Cocktail-SPK at5.9E09PFU/mL to ensure formulation is efficiently sprayed out of thecontainer closure system (i.e. dosage metered pump and 18 mL Easyfoilpouch). Stability of diluted formulation was conducted at 4° C. Table 17presents these stability results.

TABLE 17 Stability of lot F1075 stored in spray vials at 4° C. (phagetitre in PFU/mL) Microencapsulated Microencapsulated Free suspensionphages Free suspension phages Timepoint 0 2 weeks S. aureus 4.63E09 ±3.21E08 2.07E08 ± 4.79E07 2.79E09 ± 3.66E08 7.18E07 ± 2.95E07 P.aeruginosa 5.50E08 ± 6.54E08 9.96E07 ± 5.39E06 3.20E08 ± 5.29E07 1.03E08± 9.33E06 K. pneumoniae 1.28E09 ± 5.83E07 9.65E07 ± 5.39E06 1.18E09 ±2.25E08 2.74E07 ± 2.07E06 Complete 6.46E09 ± 6.06E08 4.03E08 ± 5.49E074.29E09 ± 2.53E08 2.02E08 ± 4.05E07 cocktail Timepoint 4 weeks 8 weeksS. aureus 4.13E09 ± 5.69E08 1.75E08 ± 4.70E07 7.98E08 ± 1.98E08 1.26E07± 1.45E07 P. aeruginosa 2.13E08 ± 1.53E07 1.28E08 ± 1.08E07 3.37E08 ±3.21E07 6.32E07 ± 5.06E07 K. pneumoniae 2.12E08 ± 7.49E07 2.55E07 ±4.25E06 3.65E08 ± 9.09E07 2.55E06 ± 2.45E06 Complete 4.56E09 ± 5.38E083.28E08 ± 5.19E07 1.50E09 ± 3.02E08 7.83E07 ± 6.74E07 cocktail Timepoint12 weeks 16 weeks S. aureus 1.14E08 ± 2.10E07 1.30E07 ± 1.73E07 4.14E08± 2.19E08 2.39E07 ± 1.98E07 P. aeruginosa 3.07E08 ± 4.51E07 3.52E07 ±3.77E06 3.13E08 ± 4.62E07 3.92E07 ± 1.53E07 K. pneumoniae 5.80E07 ±1.47E07 5.41E06 ± 1.84E06 9.83E07 ± 6.37E07 1.67E07 ± 7.47E06 Complete4.79E08 ± 3.21E07 5.36E07 ± 2.07E07 4.14E08 ± 2.38E08 7.97E07 ± 1.75E07cocktail Timepoint 20 weeks 24 weeks S. aureus 3.26E07 ± 1.41E07 2.50E06± 6.75E05 9.11E07 ± 1.01E08 2.09E06 ± 1.07E06 P. aeruginosa 1.80E08 ±6.93E07 5.32E07 ± 4.29E07 2.30E08 ± 2.00E07 2.89E06 ± 2.04E05 K.pneumoniae 2.48E07 ± 1.06E07 8.28E06 ± 5.26E06 5.57E07 ± 5.51E06 2.03E06± 7.42E04 Complete 2.37E08 ± 8.67E07 6.40E07 ± 4.88E07 3.77E08 ± 8.71E077.00E06 ± 9.34E05 cocktail

Example 7: Microcapsule Dimensions in Large-Scale Manufacturing

A formulation similar to formulation F9 mentioned hereinabove wasmanufactured at a larger scale. The PEAU solution was concentrated at11% instead of 13% and a rotor/stator homogenizer at 20 000 RPM was usedto form the primary emulsion and a reactor with overhead mixer at100-300 RPM was used to prepare the secondary emulsion, similarly toformulation F10′. Also, the both primary and secondary solutionsincluded 1% of PVA (w/v). The corresponding size distribution is shownin FIG. 7 .

Example 8: Microcapsule Manufactured Using Membrane EmulsificationTechnology

The Laboratory Dispersion Cell 1 (LDC-1) from Micropore Technology Inc.unit was used to manufacture microcapsules having relatively uniformdimensions. This unit uses a membrane emulsification process.

The LDC generates droplets by pumping a disperse phase (DP, in this casea primary emulsion of phage solution in polymer-DCM solution, thepolymer being the PEAU described above) through a porous stainless-steelmembrane, into a chamber containing the continuous phase (CP). A stirrersitting just above the surface of the membrane provides shear to breakoff the droplets as they pass through the membrane. The size of thepores, the injection rate of the DP and the stirrer speed can all beadjusted to form droplets ranging from 15 to 1000 μm. In this case, themembrane selected had pores of 15 μm, with a spacing of 200 μm in a ringconfiguration. This configuration means that the pores are only arrayedunderneath the edges of the stirrer, to give an even shear profileacross the pores where the shear imparted by the stirrer is the highest.The DP was added using a syringe-pump, fitted with a 25 mL syringe. TheDP was added at a rate of 1.0 mL min-1. The stirrer was set to a speedof 10V, with the vortex-breaker positioned to prevent the formed vortexfrom affecting the shear at the surface of the membrane.

The solutions were prepared as described with respect to Formulation F9described above. The emulsion chamber was filled with CP, and the DPinjection line was primed with CP to remove all air from the system TheDP emulsion was then loaded into a syringe and attached to the DPinjection line. The stirrer was then started, and the syringe pump wasactivated to start DP addition. After addition was finished, theemulsion was transferred to a beaker and stirred with low rpm magneticstirring for 18 hours.

Stable microscapsules were successfully produced. The initialdistribution was of a narrow size distribution. When the 11 mL additionwas nearly complete, the addition was stopped. The microspheres remainedstable throughout the solvent evaporation process, with shrinkagebetween the emulsion formation and the final product. The final producthad a size distribution as shown in FIG. 8 , with a mean diameter of53.12 μm and a standard deviation of 10.93 μm.

This method allows for manufacturing microcapsules with a relativelynarrow size distribution, for example with a diameter having a standarddeviation within a batch that is 20% or less of the mean diameter.

Although the present invention has been described hereinabove by way ofexemplary embodiments thereof, it will be readily appreciated that manymodifications are possible in the exemplary embodiments withoutmaterially departing from the novel teachings and advantages of thisinvention. Accordingly, the scope of the claims should not be limited bythe exemplary embodiments, but should be given the broadestinterpretation consistent with the description as a whole. The presentinvention can also be modified, without departing from the spirit andnature of the subject invention as defined in the appended claims.

What is claimed is:
 1. A composition comprising: polymer microcapsules;and active bacteriophages encapsulated in the polymer microcapsules;wherein the polymer microcapsules include an amino-acid based polymerand the active bacteriophages are dispersed in the polymermicrocapsules; wherein the amino-acid based polymer defines a hollowstructure in which a first aqueous suspension is encapsulated in atleast some of the polymer microcapsules, at least some of the activebacteriophages being suspended in the first aqueous suspension.
 2. Thecomposition as defined in claim 1, wherein at least some of the activebacteriophages are dispersed in the amino-acid based polymer.
 3. Thecomposition as defined in claim 1, wherein the first aqueous suspensionincludes polyvinyl alcohol.
 4. The composition as defined in claim 3,wherein the first aqueous suspension includes between 0.1% and 10% w/vof the polyvinyl alcohol and wherein the polyvinyl alcohol has a meanmolecular weight of between 10 kDa and 400 kDa.
 5. The composition asdefined in claim 1, wherein the polymer microcapsules are in a secondaqueous suspension, and wherein the second aqueous suspension alsoincludes polyvinyl alcohol.
 6. The composition as defined in claim 5,wherein the second aqueous suspension includes between 1% and 5% w/v ofthe polyvinyl alcohol.
 7. The composition as defined in claim 1, whereinthe polymer microcapsules have a mean size between 20 μm and 100 μm andan upper limit size of 250 μm or less.
 8. The composition as defined inclaim 1, wherein a thickness of the polymer enclosing the first aqueoussolution in the microcapsules is between 3% and 15% of a mean size ofthe microcapsules.
 9. The composition as defined in claim 1, wherein themicrocapsules further include at least one product selected from thegroup consisting of endolysins, lysostaphins, phage proteins, phageenzymatic formulations.
 10. The composition as defined in claim 1,wherein the amino-acid based polymer is selected from (1) a poly (esteramide urea) wherein at least one diol, at least one diacid, and at leastone amino acid are linked together through an ester bond, an amide bond,and a urea bond, (2) a poly (ester urethane urea) wherein at least onediol and at least one amino acid are linked together through an esterbond, a urethane bond, and a urea bond, (3) a poly (ester amide urethaneurea) wherein at least one diol, at least one diacid, and at least oneamino acid are linked together through an ester bond, an amide bond, aurethane bond, and a urea bond, (4) a poly (ester amide urethane)wherein at least one diol, at least one diacid, and at least one aminoacid are linked together through an ester bond, an amide bond, and aurethane bond, (5) a poly (ester urea) wherein at least one diol and atleast one amino acid are linked together through an ester bond and aurea bond, and (6) a poly (ester urethane) wherein at least one diol andat least one amino acid are linked together through an ester bond and aurethane bond, further wherein the at least one diol is a compound offormula: HO—R₁—OH, R₁ is chosen from C₂-C₁₂ alkylene optionallyinterrupted by at least one oxygen, C:₃-C₈ cycloalkylene, C₃-C₁₀cycloalkylalkylene,

the at least one diacid is a compound of formula:HO—(CO)—R₃—(CO)—OH, R₃ is C₂-C₁₂ alkylene, the at least one amino acidis chosen from naturally occurring amino acids and non-naturallyoccurring amino acid.
 11. The composition as defined in claim 10,wherein the amino-acid based polymer is a poly (ester amide urea)comprising the following two blocks with random distribution thereof:

wherein the ratio of l:m ranges from 0.05:0.95 to 0.95:0.05, l+m=1, R₁is chosen from C₂-C₁₂ alkylenes optionally interrupted by at least oneoxygen, C₃-C₈ cycloalkylenes, C₃-C₁₀ cycloalkylalkylenes,

R₃ is C₂-C₁₂ alkylene, R₂ and R₄ are independently chosen from the sidechains of L- and D-amino acids so that the carbon to which R₂ or R₄ isattached has L or D chirality.
 12. The composition as defined in claim11, wherein the ratio of l:m ranges from 0.25:0.75 to 0.75:0.25, l+m=1and wherein R₁ is —(CH₂)₆—, R₃ is —(CH₂)₈— and both R₂ and R₄ are theside chain of L-leucine.
 13. The composition as defined in claim 10,wherein the amino-acid based polymer has a molecular weight between 40kDa and 105 kDa.
 14. The composition as defined in claim 10, wherein theamino-acid based polymer has a molecular weight between 40 kDa and 60kDa.
 15. The composition as defined in claim 1, wherein the compositionis in liquid form.
 16. The composition as defined in claim 15, whereinthe composition has a viscosity small enough to allow pulverizationthrough a nozzle.
 17. The composition as defined in claim 16, whereinthe polymer microcapsules are suspended in a solution including apoloxamer.
 18. The composition as defined in claim 17, wherein thepoloxamer is poloxamer 407 having a mean molecular weight of between9500 kDa and 15000 kDa and in a concentration of between 10 and 30percent.
 19. The composition as defined in claim 1, wherein thecomposition is powder form.
 20. The composition as defined in claim 1,wherein the composition is in gel form.
 21. The composition as definedin claim 1, wherein the polymer microcapsules contain on average morethan 4 active bacteriophages.
 22. The composition as defined in claim 1,wherein the polymer microcapsules contain on average more than 100active bacteriophages.
 23. The composition as defined in claim 1,wherein the composition further includes active bacteriophages outsideof the polymer microcapsules.
 24. The composition as defined in claim 1,further comprising a drug selected from the set consisting ofantibiotics, pain killer and hemostatic drug.
 25. The composition asdefined in claim 1, wherein the composition has a stability such that atleast 1% of the active bacteriophages remain active after storage of thecomposition for one year at 4° C.
 26. The composition as defined inclaim 1, wherein the microcapsules have a size distribution with astandard deviation of 20% or less of the mean size.